Methods and compositions for vein harvest and autografting

ABSTRACT

The leading cause of graft failure is the subsequent development of intimal hyperplasia, which represents a response to injury that is thought to involve smooth muscle proliferation, migration, phenotypic modulation, and extracellular matrix (ECM) deposition. Surgical techniques typically employed for vein harvest—stretching the vein, placing the vein in low pH, solutions, and the use of toxic surgical skin markers—are shown here to cause injury. The invention therefore provides for non-toxic surgical markers than also protect against stretch-induced loss of functional viability, along with other additives. Devices and compositions for reducing physical stress or protecting from the effects flowing therefrom, also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/267,640, filed Dec. 8, 2009, the entire contentsof which are hereby incorporated by reference.

GOVERNMENT SUPPORT

The invention was made with government support under Grant No. 2R01HL070715 from the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the fields of autologous vein, veingraft, vein preservation, tissue preservation, intimal hyperplasia,vasospasm, pharmaceuticals, devices, and vascular biology.

BACKGROUND OF THE INVENTION

Human greater saphenous vein (HSV) remains the most commonly usedconduit for coronary and peripheral arterial bypass grafting. HSV istypically harvested from the leg with direct surgical exposure orendoscopic vein harvest. The branches are ligated and the vein isremoved and placed on the “back table” prior to implantation. Mostsurgeons place the HSV in heparinized saline solution at roomtemperature. The vein is cannulated at the distal end and manuallydistended (with a syringe) with heparinized saline. This allows foridentification and ligation of side branches that have been missedduring harvest. This manual distension leads to injury to the vein. Theveins are also marked with a surgical skin marker to optimizeorientation during implantation.

Of the more than 1 million coronary bypass procedures that areundertaken each year worldwide, 10-15% of coronary vein grafts undergoearly thrombotic occlusion; an additional 10-15% occlude in the next 1-5years due to intimal hyperplasia, with a further 30-40% occluding in thesubsequent 5-7 years because of progressive atherosclerosis superimposedon intimal hyperplasia. Less than half of vein grafts remain patentafter 12 years (Motwani & Topol, 1998). Vein graft occlusion leads tomyocardial infarction, limb loss, and death.

The leading cause of failure of arterial bypass grafts is intimalhyperplasia (Clowes & Reidy, 1991). Despite the many recenttechnological advances in vascular interventions, intimal hyperplasiaremains an expensive, morbid, and unsolved problem. Intimal hyperplasiais mediated by a sequence of events that include vascular smooth muscleproliferation, migration, phenotypic modulation, and extracellularmatrix production (Allaire & Clowes, 1997; Mosse et al., 1985). Thisprocess leads to pathologic narrowing of the vessel lumen, graftstenoses, and ultimately graft failure (LoGerfo et al., 1983).

A number of drugs that have been tested for their capacity to inhibitintimal hyperplasia have failed in clinical trials. Antithrombotic andantiplatelet agents such as warfarin, clopidogrel, and aspirin, havelittle or no effect on intimal hyperplasia (Kent & Liu, 2004). Drugeluting stents have been shown to be effective in preventing restenosisafter coronary angioplasty; however, no therapeutic has been approvedfor autologous conduits. Two large clinical trials for the prevention ofcoronary and peripheral vascular vein graft failure using an E2F decoy(a short sequence of DNA that binds to transcription factors,sequestering these proteins) to prevent smooth muscle proliferationfailed in their primary endpoint. Data from these large clinical trialssuggests that simply limiting the proliferation response is not adequateto prevent intimal hyperplasia (Mann et al., 1999; Alexander et al.,2005). Therefore mechanisms other than proliferation need to be targetedfor successful prevention of vein graft failure.

Injury to the vein graft during harvest leads to vasospasm and intimalhyperplasia, which cause the grafts to occlude. Thus, it would be ofgreat benefit to identify new surgical methods and therapeutics toprevent injury to the graft during harvest and subsequent intimalhyperplasia

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod of treating a vein explant prior to transplant comprising (a)providing a vein explant; (b) stabilizing the vein explants in abuffered solution comprising a P2X₇ receptor antagonist at a pH 7.0-7.6to produce a stabilized vein explant; and (c) preserving functionalviability of the stabilized vein explant. The method may further restorefunctional viability of the vein explant that before step (b) was notviable. Functional viability of smooth muscle is defined here as theability to contract in response to depolarization or agonists. Forendothelium, viability is defined the ability of pre-contracted vesselsto relax in response to acetylcholine. Additionally, the bufferedsolution may further comprise heparin. The P2X₇ receptor antagonist maybe erioglaucine/Blue Dye #1 or brilliant blue G, or a combination ofthese. Yet further, the buffered solution may comprise phosphatebuffered saline, MOPS, Hepes, Pipes, acetate or Plasmalyte. The pH maybe 7.35-7.45, or 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6.

Additionally, the buffered solution may further comprise magnesiumsulfate or Hanks' Balanced Salt Solution.

Additionally, the buffered solution may further comprise one or more ofan anti-contractile agent, an anti-oxidant agent, an oligosaccharide, acolloid agent, an anti-inflammatory agent, an endothelial functionpreservative, a metabolic regulator, a hydrogel, an inhibitor of heatshock protein 27 (HSP27), a regulator of HSP20, and/or an inhibitor ofMAPKAP kinase 2.

Further, the anti-contractile agent may be at least one of aphosphodiesterase inhibitor (e.g., papaverine, sildenafil, tadalafil,vardenafil, udenafil, avanafil cilistizol, pentoxifylline, dipyridamoleor a combination thereof), a calcium channel blocker (e.g., amlodipine,aranidipine, azelnidipine, barnidipine, cilnidipine, clevidipine,efonidipine, felodipine, lacidipine, lercanidipine, mandipine,nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine,netrendipine, prandipine or a combination thereof), a nitric oxide donor(e.g., sodium nitroprusside, nitroglycerin or a combination thereof), ora cyclic nucleotide analogue (dibutyryl cAMP, dibutyryl cGMP or acombination thereof), or a combination thereof.

Further, the anti-oxidant agent may be e.g., N-acetylcysteine,allopurinol, glutathione, mannitol, ascorbic acid, a tocopherol, atocotrienol or a green tea phenol or a combination thereof.

Further, the oligosaccharide may be e.g., lactobionic acid, raffinose,or trehalose or a combination thereof.

Further, the colloid agent may be, e.g., hydroxyethyl starch, dextran,blood or albumin or a combination thereof.

Further, the anti-inflammatory agent may be, e.g., a corticosteroid(e.g., dexamethasone, hydrocortisone, cortisone, prednisone,prednisolone, methylprednisolone or a combination thereof), or anonsteroidal anti-inflammatory (e.g., aspirin, ibuprophen, naproxensalicylic acid or a combination thereof), a MAPKAP kinase 2 inhibitor,anti-TNF-α, anti-IL-1-β, a Cox-2 inhibitor, or a combination thereof

Additionally, the endothelial function preservative may be, e.g., anangiotensin converting enzyme inhibitor (e.g., enalapril, ramipril,quinapril, perindopril, lisinopril, benazepril, monopril or acombination thereof), an angiotensin receptor inhibitor (e.g. losartan),a statin (e.g. atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin or acombination thereof), metformin, aminoimidazole carboxamideribonucleotide (AICAR) or an estrogen (e.g., estriol, estradiol,estrone, 17β-estradiol or a combination thereof).

Additionally, the metabolic regulator may be, e.g., glucose, adenosineamylin, calcitonin related gene peptide, insulin, or a combinationthereof.

Additionally, the hydrogel may be composed of, for example, a naturalpolysaccharide such as alginate, dextran, chitosan, andglycosaminoglycan, or a hydrophilic polymer such as polyethylene glycol,methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose,polyhydroxbuterate, or poly(n-isopropylacrylamide).

Further, the inhibitor of HSP27 may be, for example, an siRNA or miRNAthat inhibits HSP27 expression, an anti-miRNA that enhances HSP20expression, or a combination thereof.

Further, the inhibitor of MAPKAP kinase 2 may be, for example, a peptideinhibitor.

The explant may be marked with a non-alcohol based marker, such as,without limitation, erioglaucine/Blue Dye #1, indigotine, Allura Red AC,or brilliant blue G.

The method may further comprise flushing the lumen of the vein explantsuch that the internal flushing pressure does not exceed 200 mm Hg, ordoes not exceed 150 mm Hg.

In another embodiment, there is provided a vein transplant kitcomprising (a) a tissue marking pen comprising a P2X₇ receptorantagonist; and (b) a physiologic buffered solution or reagents formaking such. Additionally, the kit may further comprise a containersuitable for bathing a vein explant. Additionally, the kit may furthercomprise one or more of heparin, an anti-contractile agent, ananti-oxidant agent, an oligosaccharide, a colloid agent, ananti-inflammatory agent, an endothelial function preservative, ametabolic regulator, a hydrogel, an inhibitor of a heat shock protein,magnesium sulfate, and/or an inhibitor of MAPKAP kinase 2.

The buffered solution may comprise, for example, phosphate bufferedsaline, MOPS, Hepes, Pipes, acetate or Plasmalyte. The buffered solutionmay be at pH 7.0-7.6, or at 7.35-7.45. The P2X₇ receptor antagonist maycomprise, for example, erioglaucine/Blue Dye #1, Allura Red AC,brilliant blue G, or any combination thereof.

The kit may further comprise a device for flushing the lumen of a veinexplant; said device is designed to prevent flushing pressures insidethe vein explant of greater than 200 mm Hg, or greater than 150 mm Hg.The device may comprise a syringe and/or a catheter and a pop-off valve.Additionally, the syringe or catheter may comprise a bullet-shaped tipcomprising a lumen for introduction into a proximal end of said veinexplant. Additionally, the kit may further comprise a clamp designed tohold said vein explant.

Also provided is a device for flushing the lumen of a vein explant; saiddevice is designed to prevent flushing pressures inside the vein explantof greater than 200 mm Hg, or greater than 150 mm Hg. The device maycomprise a syringe and/or catheter and a pop-off valve. The syringe orcatheter may comprise a bullet-shaped tip comprising a lumen forintroduction into a distal end of said vein explant. Further, the devicemay further comprise a bullet-shaped plug lacking a lumen forintroduction into a proximal end of said vein explant. Additionally, thedevice may further comprise a clamp designed to hold said vein explant.

Still yet another embodiment comprises a buffered solution of pH7.0-7.6, wherein said buffered solution further comprises heparin and aP2X₇ receptor antagonist. The P2X₇ receptor antagonist may be, forexample, erioglaucine/Blue Dye #1 or brilliant blue G, or a combinationthereof. Additionally, the buffered solution may further compriseheparin, along with one or more of erioglaucine/Blue Dye #1, brilliantblue G, or both. Further, the buffered solution may comprise phosphatebuffered saline, MOPS, Hepes, Pipes, acetate or Plasmalyte. Further, thepH may be 7.35-7.45, or 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6.

Additionally, the buffered solution may further comprise magnesiumsulfate or Hanks' Balanced Salt Solution.

Additionally, the buffered solution may further comprises one or more ofan anti-contractile agent, an anti-oxidant agent, an oligosaccharide, acolloid agent, an anti-inflammatory agent, an endothelial functionpreservative, a metabolic regulator, a hydrogel, an inhibitor of heatshock protein 27 (HSP27), a regulator of HSP20, an inhibitor of MAPKAPkinase 2, and/or combinations thereof.

Further, the anti-contractile agent may be a phosphodiesterase inhibitor(e.g., papaverine, sildenafil, tadalafil, vardenafil, udenafil, avanafilcilistizol, pentoxifylline, dipyridamole or a combination thereof), acalcium channel blocker (e.g. amlodipine, aranidipine, azelnidipine,barnidipine, cilnidipine, clevidipine, efonidipine, felodipine,lacidipine, lercanidipine, mandipine, nicardipine, nifedipine,nilvadipine, nimodipine, nisoldipine, netrendipine, prandipine), anitric oxide donor (sodium nitroprusside, nitroglycerin or a combinationthereof), or a cyclic nucleotide analogue (e.g. dibutyryl cAMP,dibutyryl cGMP or a combination thereof).

Further, the anti-oxidant agent may be, e.g., N-acetylcysteine,allopurinol, glutathione, mannitol, ascorbic acid, a tocopherol, atocotrienol or a green tea phenol, or a combination thereof.

The oligosaccharide may be e.g., lactobionic acid, raffinose, trehalose,or a combination thereof.

The colloid agent may be, e.g., hydroxyethyl starch, dextran, blood oralbumin or a combination thereof.

The anti-inflammatory agent may be, e.g., a corticosteroid (e.g.dexamethasone, hydrocortisone, cortisone, prednisone, prednisolone,methylprednisolone or a combination thereof), a nonsteroidalanti-inflammatory (e.g. aspirin, ibuprophen, naproxen salicylic acid ora combination thereof), a MAPKAP kinase 2 inhibitor, anti-TNF-α,anti-IL-1-β, a Cox-2 inhibitor or a combination thereof.

Further, the endothelial function preservative may be an angiotensinconverting enzyme inhibitor (e.g., enalapril, ramipril, quinapril,perindopril, lisinopril, benazepril, monopril or a combination thereof),an angiotensin receptor inhibitor (e.g., losartan), a statin (e.g.,atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin, simvastatin or a combinationthereof), metformin, an estrogen (e.g., estriol, estradiol, estrone,17β-estradiol or a combination thereof) or a combination thereof.

Further, the metabolic regulator may be e.g., glucose, adenosine amylin,calcitonin related gene peptide, insulin or a combination thereof.

Additionally, the hydrogel may be composed of a natural polysaccharidesuch as alginate, dextran, chitosan, and glycosaminoglycan, or ahydrophilic polymer such as polyethylene glycol, methylcellulose,hydroxymethylcellulose, hydroxyethylcellulose, polyhydroxbuterate, orpoly(n-isopropylacrylamide).

Further, the inhibitor of HSP27 may be, for example, an siRNA or miRNAthat inhibits HSP27 expression, an anti-miRNA that enhances HSP20expression or a combination thereof.

The inhibitor of MAPKAP kinase 2 may be, e.g., a peptide inhibitor.

Thus, the compositions of the present invention have broad usesincluding use in healthcare by providing sterile medical devices andsurface sterilization and decontamination.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the variable smooth muscle functional viability in humansaphenous vein.

FIGS. 2A-B show that the current surgical harvest techniques lead todecreased smooth muscle functional viability.

FIG. 3 demonstrates that the current surgical harvest techniques lead toreduced endothelial functional viability.

FIGS. 4A-B show that the current surgical harvest techniques reduceendothelial-independent relaxation of human saphenous vein.

FIG. 5 demonstrates that human saphenous vein grafts with blue markingsdisplayed reduced smooth muscle functional viability.

FIG. 6 demonstrates that surgical skin marking reduced smooth muscleviability of human saphenous vein.

FIG. 7 shows surgical skin marking pens reduce the viability of pigsaphenous vein.

FIG. 8 shows functional response (contractile response to KCl)correlates with cell viability in human saphenous veins.

FIG. 9 demonstrates that erioglaucine restores functional viabilityafter stretch injury in porcine saphenous vein.

FIG. 10 shows that Allura Red did not restore stretch-induced injury inporcine saphenous veins.

FIG. 11 demonstrates that erioglaucine restores smooth muscle viabilityin human saphenous vein.

FIGS. 12A-C show that erioglaucine blocks BzATP-induced contraction insaphenous vein.

FIG. 13 demonstrates that the erioglaucine reduces intimal thickness inhuman saphenous vein in an organ culture model.

FIG. 14 shows erioglaucine reduces intimal layer thickening in distendedporcine saphenous vein.

FIG. 15 demonstrates that manipulation during surgical preparationimpair endothelial dependent relaxation in human saphenous vein.

FIG. 16 shows that a pressure release (pop-off) valve limits pressure inhuman saphenous vein during manual distention.

FIG. 17 shows that manual distension with a pressure release valveprevents loss of endothelial function in porcine saphenous vein.

FIG. 18A-B show that preincubation with papaverine inhibits histamineand KCl induced contractions in porcine coronary artery.

FIG. 19A-B show that preincubation with papaverine inhibitsnorepinephrine induced contractions in human saphenous vein.

FIG. 20 shows the vein harvest device kit.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention provides new methods and reagents with whichto harvest, treat, preserve and transplant autologous conduits andinhibit intimal hyperplasia. The pH of the solution used to storeautologous vein conduits prior to implantation, which includesheparinized saline, is highly acidic (pH 6.2). This acidic pH has beenshown to reduce the functionality of the conduit. Moreover, the use ofsurgical skin markers comprising isopropyl alcohol, to mark theautologous conduits, also reduces the functionality of the conduit.Erioglaucine, otherwise known as FD&C blue dye #1, is not toxic to thevein and restores functional integrity after injury. It also has beenshown that common manual distension of the vein can lead to intraluminalpressures greater than 300 mm Hg, which also has a deleterious effect onconduit functionality. Placing a pop off valve on the syringe reducesthe maximal possible intraluminal pressure to 130-140 mm Hg, therebyprotecting the vein conduit.

I. Harvest Solution

In one aspect, the present invention provides a buffered solution, pH7.0-7.6, in which to place the vein after harvest. In one embodiment thebuffer is phosphate buffered saline; however, MOPS, Hepes, Pipes, andacetate are alternative formulations. Magnesium sulfate (5 mM) can alsobe added to the solution to stabilize membranes.

Another buffer option is Plasma-Lyte 56 Injection (Multiple ElectrolytesInjection, Type 1, USP) a sterile, nonpyrogenic, hypotonic solution in asingle dose container for intravenous administration. Each 100 mLcontains 234 mg of Sodium Chloride, USP (NaCl); 128 mg of PotassiumAcetate, USP (C₂H₃KO₂); and 32 mg of Magnesium Acetate Tetrahydrate(Mg(C₂H₃O₂)2·4H₂O). It contains no antimicrobial agents. The pH isadjusted with hydrochloric acid.

In another aspect of the invention, the harvest solution can be preparedas a highly viscous solution such as that described in Seal & Panitch(2003). These authors described a rapidly forming polymer matrix withaffinity-based controlled release properties was developed based uponinteractions between heparin-binding peptides and heparin. Dynamicmechanical testing of 10% (w/v) compositions consisting of a 3:1 molarratio of poly(ethylene glycol)-co-peptide (approximately 18,000 g/mol)to heparin (approximately 18,000 g/mol) revealed a viscoelastic profilesimilar to that of concentrated, large molecular weight polymersolutions and melts. In addition, the biopolymer mixtures recoveredquickly following thermal denaturation and mechanical insult. Thesegel-like materials were able to sequester exogenous heparin-bindingpeptides and could release these peptides over several days at ratesdependent on relative heparin affinity. The initial release rates rangedfrom 3.3% per hour for a peptide with low heparin affinity to 0.025% perhour for a peptide with strong heparin affinity. By altering theaffinity of peptides to heparin, a series of peptides can be developedto yield a range of release profiles useful for controlled in vivodelivery of therapeutics.

II. Supplemental Solution Additives

In another aspect of the invention, the solutions of the presentinvention may contain additional additives to address various protectiveaspects of the invention.

For example, the solutions of the present invention may include heparin(1-10 U/ml) to prevent thrombus formation. Heparin is a highly sulfatedglycosaminoglycan that is widely used as an injectable anticoagulant,and has the highest negative charge density of any known biologicalmolecule. It can also be used to form an inner anticoagulant surface onvarious experimental and medical devices such as test tubes and renaldialysis machines. Pharmaceutical grade heparin is derived from mucosaltissues of slaughtered meat animals such as porcine (pig) intestine orbovine (cow) lung.

Although used principally in medicine for anticoagulation, the truephysiological role of heparin in the body remains unclear, because bloodanti-coagulation is achieved mostly by endothelial cell-derived heparansulfate proteoglycans. Heparin is usually stored within the secretorygranules of mast cells and released only into the vasculature at sitesof tissue injury. It has been proposed that, rather thananticoagulation, the main purpose of heparin is in a defensive mechanismat sites of tissue injury against invading bacteria and other foreignmaterials. In addition, it is preserved across a number of widelydifferent species, including some invertebrates that do not have asimilar blood coagulation system.

Native heparin is a polymer with a molecular weight ranging from 3 kDato 50 kDa, although the average molecular weight of most commercialheparin preparations is in the range of 12 kDa to 15 kDa. Heparin is amember of the glycosaminoglycan family of carbohydrates (defined as anorganic compound which has the empirical formula Cm(H2O)n; that is,consists only of carbon, hydrogen and oxygen, with a hydrogen:oxygenatom ratio of 2:1). Glycosaminoglycans (GAGs) or mucopolysaccharides arelong unbranched polysaccharides consisting of a repeating disaccharideunit. The repeating unit consists of a hexose (six-carbon sugar) or ahexuronic acid, linked to a hexosamine (six-carbon sugar containingnitrogen).

Heparin, (which includes the closely-related molecule heparan sulfate)consists of a variably-sulfated repeating disaccharide unit. The maindisaccharide units that occur in heparin are shown below. The mostcommon disaccharide unit is composed of a 2-O-sulfated iduronic acid and6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S). For example,this makes up 85% of heparins from beef lung and about 75% of those fromporcine intestinal mucosa. Not shown below are the rare disaccharidescontaining a 3-O-sulfated glucosamine (GlcNS(3S,6S)) or a free aminegroup (GlcNH₃ ⁺). Under physiological conditions, the ester and amidesulfate groups are deprotonated and attract positively-chargedcounterions to form a heparin salt. It is in this form that heparin isusually administered as an anticoagulant.

In another aspect, the harvest solution can be a hydrogel that coats thevessel to minimize volume while keeping the vessel moist. In addition,the hydrogel can contain a therapeutic to help maintain vasorelaxation.Hydrogels include those synthesized from hydrophilic polymers that arecrosslinked through covalent bods such as poly (ethylene glycol),polyacrylamide, polyfumerate, poly(N-siopropyl acrylamide), etc., or anygel like material crosslinking through physical interactions includinghydrophobic and ionic. Gels include polyurethanes, agarose andalginates.

In another aspect of the invention, the present invention includespapaverine (1 mM) to inhibit contraction and spasm of the vein.Alternative anti-spasmodic agents are nicardipine, sodium nitroprusside,nitroglycerine (0.5-1.0 mM), or dibutyryl cAMP (2 mM).

In another aspect of the invention, the present invention includesantioxidants to prevent oxidative damage to the vein. N-acetylcysteine(10 mM), allopurinol (1 mM), glutathione (3 mM), mannitol (30-60 mM), orgreen tea phenols (0.5-1.0 mg/ml) are particular antioxidants ofinterest.

In another aspect, the present invention provides oligosaccharides inthe harvest solution to prevent desiccation of the graft. Lactobionicacid (100 mM), raffinose (30 mM), or trehalose (30 mM) are particularoligosaccharides. Lactobionic acid is a disaccharide that providesosmotic support and prevents cell swelling. Raffinose is a trisaccharidethat provides hypertonicity. Trehalose is a disaccharide with waterretention properties.

In another aspect, the present invention provides starch in the harvestsolution to support colloid osmotic pressure. Hydroxyethyl starch (30-50mM), dextran (40 g/l), blood, or albumin, are particularly contemplatedcolloid agents.

In another aspect of the invention, the present invention includesanti-inflammatory agents. Steroids such as dexamethasone (5-10 mg/l) orsalicylic acid are examples of anti-inflammatory agents.

In another aspect of the invention, drugs will be included to preventendothelial dysfunction. Angiotensin converting enzyme inhibitors,statins, metformin, AICAR and estrogens are examples of such drugs.

In another aspect of the invention, the present invention includesmetabolic regulators. Glucose (200 mM), adenosine (5 mM), and insulin(100 U/ml) are particularly contemplated metabolic regulators.

In another aspect of the invention, the present invention includes anovel peptide inhibitor of MAPKAP kinase 2 (and related peptides) toreduce inflammation, enhance relaxation of the smooth muscle, andprevent spasm. PCT Applications US2007/16246 and US2008/72525 describesuch inhibitors, and are incorporated by reference herein.

In another aspect of the invention, the present invention includes siRNAor miRNA to decrease the expression of HSP27 to prevent intimalhyperplasia. The sense strand siRNA sequences are 1)GACCAAGGAUGGCGUGGUGUU (SEQ ID NO: 1) and 2) AUACACGCUGCCCCCCGGUUU (SEQID NO: 2). The sense strand miRNA sequences are 1) miR-580 or miR-1300,AACUCUUACUACUUAGUAAUCC (SEQ ID NO: 3) and 2) miR-552,UUGUCCACUGACCAAUCUGUU (SEQ ID NO: 4). The anti-miR-320 sequence is:UCGCCCUCUCAACCCAGCUUUU Expression of the siRNA and miRNA is plasmidbased or synthetic. Delivery of the DNA or synthetic oligo-duplexes canbe performed via reversible permeabilization or pressurization (Monahanet al., 2009).

III. P2X₇ Receptor Antagonists

Injury leads to prolonged release of ATP which can activate ATPreceptors (Khakh & North, 2006). P2X receptors are a family ofligand-gated ion channels that bind extracellular ATP. The P2X₇ receptoris responsible for the ATP-dependent lysis of macrophages and is alsofound on human saphenous vein smooth muscle (Cario-Toumaniantz et al.,1998). Activation of the P2X₇ receptor can form membrane pores permeableto large molecules in human saphenous vein (Cario-Toumaniantz et al.,1998). This leads to increases in intracellular Ca²⁺ which can activatecaspases, and ultimately lead to cell death due to autolysis andapoptosis (Donnelly-Roberts et al., 2004). Activation of the P2X₇receptor has been associated with activation of p38 MAPK pathway andchanges in the actin cytoskeleton (Pfeiffer et al., 2004). Activation ofP2X₇ receptor also leads to production and release of interleukins andother cytokines which contributes to an inflammatory response(Donnelly-Roberts et al., 2004). Recently, systemic administration of anantagonist of the P2X₇ receptor has been shown to improve recovery in arodent model of stretch induced spinal cord injury (Peng et al., 2009).

A variety of P2X₇ receptor antagonists have been described in theliterature. For example, Alcaraz et al. (2003) describe the synthesisand pharmacological evaluation of a series of potent P2X₇ receptorantagonists. The compounds inhibit BzATP-mediated pore formation inTHP-1 cells. The distribution of the P2X₇ receptor in inflammatorycells, most notably the macrophage, mast cell and lymphocyte, suggeststhat P2X₇ antagonists have a significant role to play in the treatmentof inflammatory disease. Carroll et al. (2009) review distinct chemicalseries of potent and highly selective P2X₇ receptor antagonists.

The following U.S. Patents, incorporated herein by reference, discloseP2X₇ receptor antagonists: U.S. Pat. Nos. 7,709,469, 6,812,226,7,741,493 7,718,693 and 7,326,792. The following U.S. PatentPublications, incorporated herein by reference, disclose P2X₇ receptorantagonists: 2010/0292295, 2010/0292224, 2010/0286390, 2010/0210705,2010/0168171, 2010/0160389, 2010/0160388, 2010/0160387, 2010/0160384,2010/0160373, 2010/0144829, 2010/0144727, 2010/0105068, 2010/0075968,2010/0056595, 2010/0036101, 2009/0264501, 2009/0215727, 2009/0197928,2009/0149524, 2009/0005330, 2008/0132550, 2008/0009541, 2007/0122849,2007/0082930, 2005/0054013, 2005/0026916 and 2002/0182646.

As discussed above, an aspect of the invention includes a marker thatcontains a non-toxic dye to mark the vein. FD&C Blue #1 (erioglaucine),an artificial food dye approved by the FDA (E #133), also has not onlybeen shown to be non-toxic, but protective of harvest techniques thatare injurious to saphenous veins and is a P2X₇ receptor antagonist.Brilliant blue G, an analog erioglaucine, also is contemplated as a P2X₇receptor antagonist.

Indigotine (E132) is another dark blue artificial dye approved by theFDA. Fast Green (E143) is another bluish green artificial dye approvedby the FDA. Natural dyes such as curcurmin or betanin are otheralternatives. Curcumin is the principal curcuminoid of the spice tumericand has antioxidant and anti-inflammatory properties. As a foodadditive, its E number is E100. Betanin is a red glycosidic food dyeobtained from beets and is a natural food dye. Other possible dyesinclude genestein blue, evans blue, india ink, Allura Red AC, Tartazine,and Erythrosine.

IV. Devices

Preliminary studies, discussed below, demonstrate that currently usedharvest techniques are injurious to saphenous veins. These data pose anew paradigm for thinking about vein graft failure and offer simple andstraightforward approaches to ameliorate vein graft injury.

Thus, in another aspect of the invention, the present invention includesa “pop off” valve to prevent over distension of the vein during sidebranch ligation. Qosina pressure relief T valve (part # D002501) is oneexample. In another aspect of the invention, the present inventionincludes a “bullet tipped” needle that is used to secure the vein and adevice to prevent stretch of the vein.

V. Kits

The present invention may also be embodimed in a kit for use inconjunction with surgical vein transplant procedures. Theimmunodetection kits will comprise, in suitable container means, variouscontainers, devices and/or reagents, along with appropriate instructionsfor use.

In certain embodiments, the kit will comprise harvest solutions, orreagents for making such. The solutions or reagents would be provided insterile form, optionally with sterile containers for mixing and storingharvest solutions. The kit may also advantageously comprise a chamberfor bathing/storing transplant tissue following explant and prior totransplant. Various other supplemental additives described above mayalso be included.

Another element of the kit may be the inclusion of a surgical markingpen comprising a non-toxin dye/marker, as described above. The pen maybe “preloaded” with the marker/dye, or may be provided empty, with themarker/dye in solution or in reagent form to be loaded into the pen bythe user.

Further devices including a syringe, catheter, and/or tubing equipped orincluding a pop-off valve as described above. Also included may be adevice for holding a vein in place, such as a clamp, optionally providedwith a stand or base, permitting “hands-free” positioning of the graftfor further treatment.

The container aspect of the kit will generally include means for holdingat least one vial, test tube, flask, bottle, packet, syringe, catheteror other container in a secure and protected fashion, for example, inclose confinement for commercial sale. Such means may include injectionor blow-molded plastic containers into which the desired containers,devices or reagents are retained.

VI. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Toxicity of Surgical Marking Pens to Vein Tissue

De-identified discarded segments of human saphenous vein were collected(n=66), after informed consent approved by the Institutional ReviewBoard of the Vanderbilt University (Nashville, Tenn.), from patientsundergoing coronary artery bypass or peripheral vascular bypass surgery.The veins were stored in a saline solution until the end of the surgicalprocedure at which time they were placed in cold transplant harvestbuffer (100 mM potassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄, 30 mMraffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 50 g/Lhydroxyethyl starch, pH 7.4) and stored at 4° C. The vessels were testedwithin 24 hours of harvest. The presense of blue markings were assessedfor each HSV. Rings 1.0 mm in width were cut from segments of saphenousvein dissected free of fat and connective tissue, stripped of theendothelium and were suspended in a muscle bath containing a bicarbonatebuffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO₄, 1.0 mM NaH₂PO₄, 10 mMglucose, 1.5 mM CaCl₂, and 25 mM Na₂HCO₃, pH 7.4), gassed with 95% O₂/5%CO₂ at 37° C. The rings were manually stretched to 4 g of tension, andwas maintained at a resting tension of 1 g was obtained andequilibtrated for ˜2 hr. Force measurements were obtained using aRadnoti Glass Technology (Monrovia, Calif.) force transducer (159901A)interfaced with a Powerlab data acquisition system and Chart software(AD Instruments, Colorado Springs, Colo.). To determine viability, therings were contracted with 110 mM KCl (with equimolar replacement ofNaCl in bicarbonate buffer), and the force generated was measured. Forcewas converted to stress ([Newtons (N)/m²]=force (g)×0.0987/area, wherearea is equal to the wet weight [mg/length (mm at maximal length)]divided by 1.055) 10⁵ N/m². There was variability in the functionalviability of the veins (FIG. 1). Veins generating stress of ≦0.025×10⁵N/m² were considered non-viable (grey) and those generating stress of>0.025×10⁵N/m² were viable (black). 40% of the vein tested wasnon-viable. Each point represents a different patient and an aggregateof at least three separate rings from that patient.

Segments of human saphenous vein (n=8) were collected prior topreparation of the vein for transplantation into the arterialcirculation (unmanipulated, UM) and after surgical preparation (aftermanipulation, AM). Preparation involves manual distension of the vein,marking with a surgical skin marker, and placing the vein in heparinizedsaline. The contractile response to 110 mM KCl (FIG. 2A) orphenylephrine (10⁻⁶M, FIG. 2B) was determined and force generated wasconverted to stress (10⁵ N/m²). Manipulation during vein preparation ledto decreased contractile response to KCl and phenylephrine (FIGS. 2A-B).Each point represents a different patient and an aggregate of theresponse of at least three separate rings from each patient.

Human saphenous veins were also precontracted with phenylephrine (10⁻⁶M)followed by treatment with carbachol (5×10⁻⁷M) to determine endothelialdependent relaxation (Furchgott et al., 1980). Segments of humansaphenous vein (n=5) were collected prior to preparation of the vein fortransplantation into the arterial circulation (unmanipulated, UM) andafter surgical preparation (after manipulation, AM). Rings from eachsegment were suspended in a muscle bath, equilibrated in a bicarbonatebuffer, and contracted with 110 mM KCl. After an additional 30 minequilibration in a bicarbonate buffer, rings were pre-contracted with10⁻⁶M phenylephrine (PE) and treated with 5×10⁻⁷M carbachol. Force wasmeasured and converted to stress 10⁵ N/m². Responses were expressed as %of maximum PE-induced contraction. Typical manipulation during surgicalpreparation led to reduced endothelial-dependent relaxation (FIG. 3). UMveins had 28.74±3.542% endothelial-dependent relaxation whereas AMcontracted in response to carbachol (−5.976±0.9172%).

Human saphenous veins were also precontracted with phenylephrine (10⁻⁶M) followed by treatment with sodium nitroprusside (10⁻⁷M) to determineendothelial independent relaxation. Segments of saphenous vein (n=6)were collected prior to harvest preparation (unmanipulated, UM) or afterharvest preparation (after manipulation, AM). Rings from each segmentwere suspended in a muscle bath, equilibrated in a bicarbonate buffer,and contracted with 110 mM KCl. After an additional 30 min equilibrationin a bicarbonate buffer, rings were pre-contracted with 10⁻⁶Mphenylephrine (PE) and treated with 10⁻⁷M sodium nitroprusside. Typicalmanipulation during surgical preparation reduced endothelial-independentrelaxation of human saphenous vein (FIGS. 4A-B). Representative forcetracings of the UM and AM segments collected from the same patient inresponse to PE and SNP (FIG. 4A). The endothelial independent relaxationdisplayed by the two groups, expressed as % of maximum PE-inducedcontraction, were significantly different. UM veins displayed an86.62+/−5.986% relaxation, whereas AM veins displayed a 4.292+/−1.397%relaxation (FIG. 4B).

Of the 38 veins collected from patients undergoing coronary arterybypass or peripheral vascular revascularization surgery, 16 of the veinsdid not have any visible color by surgical marking pen whereas 22 of theveins had visible color. Rings were cut from the veins, suspended in amuscle bath and equilibrated in bicarbonate buffer. The rings werecontracted with 110 mM KCl and force generated was converted to stress(10⁵ N/m²). The force generated by the two groups of veins weresignificantly different (FIG. 5). Veins that had visible blue markingdisplayed less contractile responses (0.047±0.014 10⁵N/m²) than veinsthat had no visible marking (0.174±0.023 10⁵N/m²).

De-identified discarded segments of human saphenous vein that did nothave any color were used to test the effect of different markingmethods. Rings cut from the segments were either left unmarked (control;n=12), marked with a surgical skin marker (Cardinal Health, #5227 violetmarking ink; n=5), marked in 50% isopropyl alcohol, a solvent used inthe skin marker (n=4), or marked with methylene blue (Akorn, Inc., LakeForest Ill.; n=10) and incubated for 15 min at room temperature. Therings were stripped of the endothelium and were suspended in a musclebath containing a bicarbonate buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mMMgSO₄, 1.0 mM NaH₂PO₄, 10 mM glucose, 1.5 mM CaCl₂, and 25 mM Na₂HCO₃,pH 7.4), gassed with 95% O₂/5% CO₂ at 37° C. The rings were manuallystretched to 4 g of tension, and were maintained at a resting tension of1 g and equilibtrated for ˜2 hr. Force measurements were obtained usinga Radnoti Glass Technology (Monrovia, Calif.) force transducer (159901A)interfaced with a Powerlab data acquisition system and Chart software(AD Instruments, Colorado Springs, Colo.). The rings were contractedwith 110 mM KCl (with equimolar replacement of NaCl in bicarbonatebuffer), and the force generated was converted to stress 10⁵N/m². Thethree marked groups were significantly different from the controlunmarked group (p≦0.05) (FIG. 6). The rings that did not have markingshad an average stress of 0.110±0.014 10⁵N/m², the rings that were markedwith the surgical skin marker had an average stress of 0.003±0.00110⁵N/m², rings marked with 50% isopropyl alcohol had an average stress of0.005±0.003 10⁵N/m², and rings marked with methylene blue had an averagestress of 0.014±0.01 10⁵N/m².

Freshly isolated porcine saphenous veins were used to test the effect ofdifferent marking methods. The veins were collected and placed in coldtransplant harvest buffer [100 mM potassium lactobionate, 25 mM KH₂PO₄,5 mM MgSO₄, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mMallopurinol, 50 g/L hydroxyethyl starch, pH 7.4]. The vessels werestored in transplant harvest buffer at 4° C. and tested within 24 hoursof harvest and. To test the viability, rings 1.0 mm in width were cutfrom segments of saphenous vein and dissected free of fat and connectivetissue. Saphenous vein rings were untreated (Control; n=6), marked withthe surgical skin marker (n=3), or 50% isopropyl alcohol (the solventused in the surgical marker; n=3) and incubated for 15 min at roomtemperature. The rings were then equilibrated in a muscle bath,contracted with KCl, and force was measured and converted to stress (10⁵N/m²). The rings that did not have markings had an average stress of0.263±0.039 N/m², the rings that were marked with the surgical skinmarker had an average stress of 0.114±0.017 N/m², and rings marked with50% isopropyl alcohol had an average stress of 0.00005±0.00005 N/m². Thetwo marked groups were significantly different from the control unmarkedgroup (p≦0.05). (FIG. 7).

Example 2 Live Vein Cells Correlate with Functional Viability

A live cell assay was used to determined cellular viability of humansaphenous vein. De-identified discarded segments of saphenous vein(n=13) were collected, after informed consent approved by theInstitutional Review Board of the Vanderbilt University (Nashville,Tenn.), from patients undergoing coronary artery bypass or peripheralvascular bypass surgery. The veins were stored in a saline solutionuntil the end of the surgical procedure at which time they were placedin cold transplant harvest buffer (100 mM potassium lactobionate, 25 mMKH₂PO₄, 5 mM MgSO₄, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1mM allopurinol, 50 g/L hydroxyethyl starch, pH 7.4). The vessels werestored in transplant harvest buffer at 4° C. and tested within 24 hoursof harvest. Each vein was subject to physiologic experiment and livecell assay using3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).To test the viability, rings 1.0 mm in width were cut from segments ofsaphenous vein dissected free of fat and connective tissue, some werestripped of the endothelium and suspended in a muscle bath containing abicarbonate buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO₄, 1.0 mMNaH₂PO₄, 10 mM glucose, 1.5 mM CaCl₂, and 25 mM Na₂HCO₃, pH 7.4), gassedwith 95% O₂/5% CO₂ at 37° C. The rings were manually stretched to 4 g oftension, and was maintained at a resting tension of 1 g was obtained andequilibtrated for ˜2 hr. Force measurements were obtained using aRadnoti Glass Technology (Monrovia, Calif.) force transducer (159901A)interfaced with a Powerlab data acquisition system and Chart software(AD Instruments, Colorado Springs, Colo.). The rings were contractedwith 110 mM KCl (with equimolar replacement of NaCl in bicarbonatebuffer), and the force generated was measured. Any tissue failing tocontract with KCl was considered non-viable. Force was converted tostress 10⁵ N/m² for each ring and was averaged for each vein. To assesscellular viability, three rings from each vein were placed separately in0.25 ml of 0.1% MTT solution (prepared in Dulbecco phosphate bufferedsaline, pH 7.4). For negative control, one ring was placed in 20 ml ofwater and microwaved for 10 min to inactivate any enzymatic activitybefore placing in the 0.1% MTT solution. The rings were incubated at 37°C. for 1 hr. The reaction was stopped by placing the rings in distilledwater. The tissues was weighed and placed in 1 ml of CelloSolve (Sigma)for 4 hours at 37° C. to extract the formazan pigment each. Theconcentration of the pigment was measured at 570 nm using aspectrophotometer (Beckman Coulter). The absorbance of the negativecontrol was subtracted from each sample. The viability index wasexpressed as OD₅₇₀/mg/ml. The average for each vein was calculated fromthe three rings. The average stress obtained from each vein was thenplotted against the average viability index.

The data depict a significant slope showing that there was aproportional relationship (R²=0.7262) between mitochondrial viabilityand the stress viability determined by the 110 mM KCl inducedcontraction (FIG. 8). Representative HSV rings of low (left) and high(right) viability index are shown in the inset.

Example 3 Vein Harvest Solutions and Procedures

Freshly isolated porcine saphenous vein was collected in cold transplantharvest buffer (100 mM potassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄,30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 50g/L hydroxyethyl starch, pH 7.4). The vessels were tested within 24hours of harvest and storage in transplant harvest buffer at 4° C. Thevein was dissected free of fat and connective tissue and cut into 2 cmlong segments. The segments were stretched to twice their resting length(stretched; n=7) or not manipulated (control; n=12). After stretching,the segments from both groups were further divided. A solution oferioglaucine (FCF, 2.6 mM, in 5% propylene glycol and water) or vehiclewas then applied with a cotton swab in a longitudinal line to theuntreated (FCF; n=8) or the stretched (Stretched+FCF; n=3) veinsegments. The segments were incubated at room temperature for 15 min inPlasmalyte and then cut into rings. The rings were suspended in a musclebath containing a bicarbonate buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mMMgSO₄, 1.0 mM NaH₂PO₄, 10 mM glucose, 1.5 mM CaCl₂, and 25 mM Na₂HCO₃,pH 7.4), bubbled with 95% O₂/5% CO₂ at 37° C. The rings were manuallystretched to 4 g of tension, and maintained at a resting tension of 1 gand equilibtrated for ˜2 hr. Force measurements were obtained using aRadnoti Glass Technology (Monrovia, Calif.) force transducer (159901A)interfaced with a Powerlab data acquisition system and Chart software(AD Instruments, Colorado Springs, Colo. The rings were contracted with110 mM KCl (with equimolar replacement of NaCl in bicarbonate buffer),and the force generated was converted to stress 10⁵ N/m² (FIG. 9). Thecontrol rings had an average stress of 0.47±0.034 N/m², the rings thatwere marked with the erioglaucine dye had an average stress of0.566±0.064 N/m², and rings stretched had an average stress of0.367±0.042 N/m² and the stretched rings with erioglaucine dye had anaverage stress of 0.713±0.111 N/m². The stress for the stretched veinwas significantly (*p<0.05) different from the control unstretched veinsand the stretched vein with erioglaucine dye was significantly (#p<0.05)different when compared to stretched without erioglaucine dye (FIG. 9).

However, treatment with another dye, Allura Red, did not restorefunctional viability after stretch injury of porcine saphenous vein(FIG. 10), Porcine saphenous veins (n=4) were left untreated (Control)or stretched to twice their resting length (no dye), cut into rings andsuspended in the bicarbonate buffer in a muscle bath. Rings fromstretched segments were either incubated with 50 μM Allura Red (+Red) or50 μM of erioglaucine (+FCF) for 30 min. The rings were then allowed toequilibrate in the bicarbonate buffer for before contracting with 110 mMKCl. Force generated was converted to stress (10⁵N/m²). Data representrelative contractile response to Control rings which was set as 100%.The stress for the stretched vein was not significantly different fromthe stretched vein with Allura Red (NS). Erioglaucine significantlyrestored contractile response in the stretched vein (#p≦0.05) whencompared to the stretched vein with Allura Red.

Effect of erioglaucine on human saphenous vein was determined usingde-identified discarded segments of human saphenous vein from patientsundergoing coronary artery bypass or peripheral vascular bypass surgery(n=4). The veins were stored in a saline solution until the end of thesurgical procedure at which time they were placed in cold transplantharvest buffer (100 mM potassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄,30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 50g/L hydroxyethyl starch, pH 7.4). The vessels were tested within 24 hrsof harvest storage in transplant harvest buffer at 4° C. To test theviability, rings 1.0 mm in width were cut from segments of saphenousvein dissected free of fat and connective tissue, treated with either asolution of erioglaucine (FCF, 2.6 mM, in 5% propylene glycol and water)or vehicle and incubated for 30 min at room temperature. The tissueswere then stripped of the endothelium and suspended in a muscle bathcontaining a bicarbonate buffer, gassed with 95% O₂/5% CO₂ at 37° C. Therings were manually stretched to 4 g of tension, and was maintained at aresting tension of 1 g was obtained and equilibrated for ˜2 hr. Forcemeasurements were obtained using a Radnoti Glass Technology (Monrovia,Calif.) force transducer (159901A) interfaced with a Powerlab dataacquisition system and Chart software (AD Instruments, Colorado Springs,Colo.). The rings were contracted with 110 mM KCl (with equimolarreplacement of NaCl in bicarbonate buffer), and the force generated wasmeasured. Force was converted to stress 10⁵ N/m², and was plotted forvehicle and erioglaucine rings. Representative force tracings of ringsleft untreated (control) or treated with the erioglaucine dye (FCF) aredepicted (FIG. 11A). The vehicle rings had an average stress of0.015±0.012 N/m², and the erioglaucine-treated rings had an averagestress of 0.103±0.021 N/m² (FIG. 11B). The two groups were significantlydifferent (p≦0.05).

Human saphenous vein segments were collected after harvest beforesurgical manipulation from patients undergoing coronary artery bypass orperipheral vascular bypass surgery and stored in PlasmaLyte. The vesselswere tested within 2 hours of harvest. Freshly isolated porcinesaphenous vein was collected in cold transplant harvest buffer (100 mMpotassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄, 30 mM Raffinose, 5 mMAdenosine, 3 mM Glutathione, 1 mM Allopurinol, 50 g/L Hydroxyethylstarch, pH 7.4). The vessels were tested within 24 hours of harvest.Rings 1.0 mm in width were cut from porcine saphenous veins (FIG. 12A,n=2) and unmanipulated human saphenous vein (FIG. 12B, n=4) dissectedfree of fat and connective tissue. The rings were then stripped of theendothelium and suspended in a muscle bath containing a bicarbonatebuffer, bubbled with 95% O₂/5% CO₂ at 37° C. The rings were manuallystretched to 4 g of tension, and was maintained at a resting tension of1 g was obtained and equilibrated for ˜2 hr. Force measurements wereobtained using a Radnoti Glass Technology (Monrovia, Calif.) forcetransducer (159901A) interfaced with a Powerlab data acquisition systemand Chart software (AD Instruments, Colorado Springs, Colo.). Rings werecontracted with 110 mM KCl. After an additional 30 min equilibration,rings were treated with either a solution of erioglaucine (FCF, 50-200μM for 30 minutes) or vehicle for 30 min and then contracted with 100 μMBzATP. Force was measured and converted to stress. Response wasexpressed as % of maximal KCl contraction. Representative force tracingof human saphenous vein contracted with BzATP after pretreatment withvehicle (control) or 50 μM erioglaucine (FCF pretreatment) are depictedin FIG. 12C. Pretreatment with erioglaucine (FCF) but not the vehicle(Control) significantly inhibited BzATP induced contraction (*p<0.05)(FIG. 12B).

Segments of human saphenous vein were collected prior to preparation ofthe vein for transplantation into the arterial circulation(unmanipulated, UM) and after surgical preparation (after manipulation,AM) from the same patients in PlasmaLyte and were used within 2 hr ofharvest. The segment was cut into ˜1 mm rings and one ring from eachgroup was fixed in formalin (Pre-culture). The other rings were culturedin RPMI medium supplemented with 1% L-glutamine, 1%penicillin/streptomycin and 30% fetal bovine serum at 5% CO₂ and 37° C.in the absence (Control) or presence of 50 μM erioglaucine (FCF) for 14days. After 14 days, the rings were fixed in formalin, sectioned at 5 μmand stained using Verhoff Van Gieson stain. Light micrograph of therings was captured using an Axiovert 200 and intimal thickness wasmeasured using AxioVision. Data represent % increase of intimalthickness related to basal intimal thickness of the pre-culture ringwhich was set as 0%. The error bars show the standard error of the mean.Manipulation during vein preparation increased the thickening of theintimal layer (#p=0.053 in paired t-test) and treatment witherioglaucine significantly (*p<0.05) inhibited the development ofintimal thickness when compared to Control (FIG. 13).

Fresh porcine saphenous vein was harvested by a no touch method understerile conditions and stored in cold transplant harvest buffer (100 mMpotassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄, 30 mM Raffinose, 5 mMAdenosine, 3 mM Glutathione, 1 mM Allopurinol, 50 g/L Hydroxyethylstarch, pH 7.4). The vessels were used within 24 hr of harvest. Theveins were divided into three segments that were left untreated(Unmanipulated, n=7), distended (Distended, n=8) to >300 mm Hg, ordistended in the presence of the pressure relief valve (Pop Off, n=7).Each segment was then cut into ˜1 mm rings and one ring from eachcondition was immediately fixed in formalin (Pre-culture). The otherrings were cultured in RPMI medium supplemented with 1% L-glutamine,penicillin/streptomycin and 30% Fetal bovine serum at 5% CO₂ and 37° C.in the absence (Control) or presence of either 50 μM erioglaucine (FCF),50 μM brilliant blue G (BBG) or 50 μM Allura Red (Red) for 14 days.After 14 days, the rings were fixed in formalin, sectioned at 5 μm andstained using Verhoff Van Gieson stain. Light micrograph of the ringswas captured using a Axiovert 200 and intimal thickness was measuredusing AxioVision. Treatment with erioglaucine but not allura redinhibited distension induced increases in intimal thickening, * p<0.05compared to Distended-Control (FIG. 14).

Rings of human left internal mammary artery (LIMA; n=3) and saphenousveins were obtained prior to preparation of the vein for transplantationinto the arterial circulation (unmanipulated, UM; n=5) and aftersurgical preparation (after manipulation, AM; n=5). Rings cut from theUM segments were incubated in University of Wisconsin Solution (UW),heparinized saline (HS), heparinized PlasmaLyte (HP) or heparinizedPlasmaLyte containing 30 mM trehalose (HPT) for 2 hrs at roomtemperature. Rings were cut from the veins, suspended in a muscle bathand equilibrated in bicarbonate buffer. The rings were pre-contractedwith 10⁻⁶ M phenylephrine and then treated with 5×10⁻⁷ M carbachol todetermine endothelial dependent relaxation. Rings from the LIMA hadgreater endothelial dependent relaxation than saphenous vein (FIG. 15).Manipulation during surgical preparation led to decreased endothelialdependent relaxation (FIG. 15). Storage in heparinized saline [* p<0.05compared to HS for all UM groups (UM, UW, HP, & HPT)], but not inheparinized plasmalyte, heparinized plasmalyte plus trehalose ortransplant harvest solution led to decreased endothelial dependentrelaxation (FIG. 15). Data is presented as % relaxation (compared to themaximal phenylephrine induced contraction).

De-identified discarded segments of human saphenous vein (n=5) werecollected, after informed consent approved by the Institutional ReviewBoard of the Vanderbilt University (Nashville, Tenn.), from patientsundergoing coronary artery bypass or peripheral vascular bypass surgery.The veins were stored in a saline solution until the end of the surgicalprocedure at which time they were placed in cold transplant harvestbuffer (100 mM potassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄, 30 mMraffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 50 g/Lhydroxyethyl starch, pH 7.4). The vessels were tested within 24 hours ofharvest and storage in transplant harvest buffer at 4° C. A pop offvalve was connected to a syringe at one end and to a cannulatedsaphenous vein at the other. The distal end of the saphenous vein wasalso cannulated and connected to a pressure transducer. Pressure wasmonitored while the vein was distended with a hand held syringe with andwithout the pressure release valve. The pressure monitor would notmeasure pressures above 300 mmHg. This created three groups and theywere the following: pop-off pressure (Popoff), max pressure with pop-offvalve (Max with valve), and max pressure without pop-off valve (Maxwithout valve). The veins that had a pop-off valve had a mean pressureof 129±1.265 mm Hg and maximum pressure of 141.8±1.985 mm Hg, while theveins with out the pop off valve had a maximum pressure of 300±0.00 mmHg (FIG. 16). The average and maximum pressure in the veins with thepop-off valve were significantly different from the veins without thepop-off valve (p≦0.05).

Fresh porcine saphenous vein was harvested by a no touch method understerile conditions and stored in cold transplant harvest buffer (100 mMpotassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄, 30 mM Raffinose, 5 mMAdenosine, 3 mM Glutathione, 1 mM Allopurinol, 50 g/L Hydroxyethylstarch, pH 7.4). The vessels were used within 24 h of harvest. Veins(n=4) were manually distended with a syringe in the absence (Distended)or presence of an in line pressure release valve (pop-off). Controlsegments were not distended (ND). After distension, rings were cut fromthe segments and suspended in a muscle bath. The rings wereprecontracted with 10⁻⁶ M phenylephrine and then treated with 5×10⁻⁷ Mcarbachol to determine endothelial dependent relaxation. Data ispresented as the % relaxation (compared to the maximal phenylephrineinduced contraction). Manual distension with a hand held syringe led tosignificant decreases (p<0.0005) in endothelial dependent relaxation andthe pressure release valve prevents this loss of endothelial dependentrelaxation (FIG. 17).

Porcine coronary arteries were freshly isolated from euthanized pigs andplaced directly into cold transplant harvest buffer (100 mM potassiumlactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄, 30 mM Raffinose, 5 mM Adenosine,3 mM Glutathione, 1 mM Allopurinol, 50 g/L Hydroxyethyl starch, pH 7.4).Coronary arteries were dissected free of fat and adventitial tissues andthe endothelium was removed. Transverse rings (1.0 mm thickness) werecut and suspended in muscle bath, via silk 3-0 linked to forcetransducers (Kent Scientific, CT) interfaced with a Data Translation A-Dboard (Data Translation, MA). Data was acquired with the Power Labsoftware program. Porcine coronary artery rings were suspended in amuscle bath and equilibrated in Krebs Ringer bicarbonate buffer for 2 h.The rings were stretched and the length progressively adjusted untilmaximal tension was obtained. The rings were contracted with 110 mM KCl(with equimolar replacement of NaCl in bicarbonate buffer), and theforce generated was measured and converted to stress [Newtons(N)/m²]=force (g)×0.0987/area, where area is equal to the wet weight[mg/length (mm at maximal length)] divided by 1.055. Rings were washedand equilibrated for another 30 mins. Rings were treated with 5 μMhistamine, 110 mM KCl, 1 mM papaverine (PAP), 1 mM papaverine for 10 minfollowed by 5 μM histamine or 1 mM papaverine for 10 min followed by 110mM KCl and force generated was measured- and converted to stress.Representative force tracings of rings treated with 5 μM histamine(Hist), 110 mM KCl (KCl), 1 mM papaverine (PAP), 1 mM papaverine for 10min followed by 5 μM histamine (Pap+Hist) or 1 mM papaverine for 10 minfollowed by 110 mM KCl (Pap+KCl) were depicted in FIG. 18A. Decrease instress was converted to a percentage of the maximal initial KClcontraction which was set as 100%. Papaverine treatment reduced basaltension in the rings (−15.0±3.135%) (FIG. 18B). Pretreatment of ringswith papaverine completely inhibited histamine (−12.0±4.163 compared to98.613±11.049) and KCl (−20.0±10.00 compared to 103.33±2.404%) inducedcontraction (FIG. 18B).

De-identified discarded segments of human saphenous vein (n=6) werecollected, after informed consent approved by the Institutional ReviewBoard of the Vanderbilt University (Nashville, Tenn.), from patientsundergoing coronary artery bypass or peripheral vascular bypass surgery.The veins were stored in a saline solution until the end of the surgicalprocedure at which time they were placed in cold transplant harvestbuffer (100 mM potassium lactobionate, 25 mM KH₂PO₄, 5 mM MgSO₄, 30 mMraffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 50 g/Lhydroxyethyl starch, pH 7.4). The vessels were tested within 24 hrs ofharvest and storage in transplant harvest buffer at 4° C. Veins werecleaned off fat and adventitial tissues and the endothelium was removed.Transverse rings (1.0 mm thickness) were cut and suspended in musclebath, via silk 3-0 linked to force transducers (Kent Scientific, CT)interfaced with Powerlab data acquisition system and Chart software (ADInstruments, Colorado Springs, Colo.). Human saphenous vein rings weresuspended in a muscle bath and equilibrated in Krebs Ringer bicarbonatebuffer for 2 hr. The rings were stretched and the length progressivelyadjusted until maximal tension was obtained. The rings were contractedwith 110 mM KCl (with equimolar replacement of NaCl in bicarbonatebuffer), and the force generated was measured and converted to stress[Newtons (N)/m²]=force (g)×0.0987/area, where area is equal to the wetweight [mg/length (mm at maximal length)] divided by 1.055. Rings werewashed and equilibrated for another 30 mins. Rings were treated with 0.5μM norepinephrine (NE), 1 mM papaverine (Pap), or 1 mM papaverine for 10min followed by 0.5 μM NE and force generated was measured and convertedto stress. Decrease in stress was converted to a percentage of themaximal initial KCl contraction which was set as 100%. Representativeforce tracings of rings treated with 0.5 μM NE (NE), 1 mM papaverine(Pap), or 1 mM papaverine for 10 min followed by 0.5 μM NE were depictedin FIG. 19A. Decrease in stress was converted to a percentage of themaximal initial KCl contraction which was set as 100%. n=6. Papaverinetreatment reduced basal tension in the rings (−9.772.0±3.226%).Pretreatment of human saphenous vein with papaverine completelyinhibited NE (−3.210±5.119 compared to 89.935±18.344%) inducedcontraction (FIG. 19B).

Vein harvest device is shown in FIG. 20. The distal end of the vein (thevein is reversed because of valves in the vein) is cannulated with abullet tipped plastic catheter which has a lumen for irrigation andsecured to the catheter with a spring loaded clamp. The catheter isclipped into the base. An additional bullet tipped catheter with nolumen is attached to the proximal end of the vein clipped into theopposite end of the base. The device is ratcheted open until the vein isat the same length as in vivo. A syringe with extension tubing and an inline pressure release valve is attached to the distal end of the vein.The vein can now be distended and side branches ligated.

Example 4 Prophetic Clinical Protocol

The greater saphenous vein will be surgically harvested using standardsurgical technique. The distal end of the vein will be cannulated with abullet tipped vein cannula and secured with either a clamp or a silktie. The pressure release valve will be attached to the cannula with a10 or 20 cc syringe attached to the other end of the valve. In somecases, extension tubing will be placed between the syringe and thevalve. The vein will be distended with the vein harvest solution andtributaries identified and ligated with either silk ties or clips. Thevein will be marked with the marker in the kit along one long surface tomaintain orientation of the vein. In some cases, the vein may be markedprior to removal from the patient. The vein will then be placed in theharvest solution until implanted into the arterial circulation. In oneembodiment, the dye from the pen will contain a P2X₇ receptorantagonist, and the harvest solution will not contain a P2X₇ receptorantagonist. In another embodiment, the dye from the pen will not containa P2X₇ receptor antagonist, but the solution will. In a thirdembodiment, both the pen dye and the solution will contain a P2X₇receptor antagonist.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

VII. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating a vein explant prior totransplant comprising: (a) providing a vein explant; (b) stabilizing thevein explant in a buffered solution comprising a P2X₇ receptorantagonist at a pH 7.0-7.6 to produce a stabilized vein explant; and (c)preserving functional viability of the stabilized vein explant.
 2. Themethod of claim 1, wherein the method restores functional viability ofthe vein explant that, before step (b), was not viable.
 3. The method ofclaim 1, wherein said buffered solution further comprises heparin. 4.The method of claim 2, wherein said buffered solution comprises (i)heparin and (ii) erioglaucine/Blue Dye #1 and/or brilliant blue G. 5.The method of claim 1, wherein said buffered solution comprisesphosphate buffered saline, MOPS, Hepes, Pipes, acetate or Plasmalyte. 6.The method of any one of claims 1-5, wherein said buffered solutionfurther comprises one or more of an anti-contractile agent, ananti-oxidant agent, an oligosaccharide, a colloid agent, ananti-inflammatory agent, an endothelial function preservative, ametabolic regulator, a hydrogel, an inhibitor of heat shock protein 27(HSP27), a regulator of HSP20, and/or an inhibitor of MAPKAP kinase 2.7. The method of claim 6, wherein said anti-contractile agent is aphosphodiesterase inhibitor, a calcium channel blocker, a nitric oxidedonor, or a cyclic nucleotide analog.
 8. The method of claim 6, whereinsaid anti-oxidant agent is N-acetylcysteine, allopurinol, glutathione,mannitol, ascorbic acid, a tocopherol, a tocotrienol or a green teaphenol.
 9. The method of claim 6, wherein said oligosaccharide islactobionic acid, raffinose, or trehalose.
 10. The method of claim 6,wherein said colloid agent is hydroxyethyl starch, dextran, blood oralbumin.
 11. The method of claim 6, wherein said anti-inflammatory agentis a corticosteroid, a nonsteroidal anti-inflammatory, a mapkap kinase 2inhibitor, anti-TNF-α, anti-IL-1-β, or a Cox-2 inhibitor.
 12. The methodof claim 6, wherein said endothelial function preservative is anangiotensin converting enzyme inhibitor, an angiotensin receptorinhibitor, a statin, metformin or an estrogen.
 13. The method of claim6, wherein said metabolic regulator is glucose, adenosine amylin,calcitonin related gene peptide, or insulin.
 14. The method of claim 6,wherein said hydrogel is composed of a natural polysaccharde such asalginate, dextran, chitosan, and glycosaminoglycan, or a hydrophilicpolymer such as polyethylene glycol, methylcellulose,hydroxymethylcellulose, hydroxyethylcellulose, polyhydroxbuterate, orpoly(n-isopropylacrylamide).
 15. The method of claim 6, wherein saidinhibitor of HSP27 is an siRNA or miRNA that inhibits HSP27 expression.16. The method of claim 6, wherein said regulator of HSP20 is ananti-miRNA that enhances HSP20 expression.
 17. The method of claim 6,wherein said inhibitor of MAPKAP kinase 2 is a peptide inhibitor. 18.The method of claim 1, wherein said explant is marked with a non-alcoholbased marker.
 19. The method of claim 18, wherein said P2X₇ receptorantagonist is erioglaucine/Blue Dye #1 or brilliant blue G.
 20. Themethod according to claim 19, wherein the method restores functionalviability of a vein explant that, before stabilizing step (b), was notviable.
 21. The method of claim 1, wherein the stabilized vein explantis further characterized as being capable of generating a contractileforce greater than a control blood vessel when contracted with KCl invitro.
 22. The method of claim 1, wherein the stabilized vein explant isfurther characterized as being capable of withstanding inside flushingpressures of less than 200 mm Hg when distended.
 23. The method of claim22, wherein the lumen of the stabilized vein explants is flushed withthe buffered solution at flushing pressures of less than 200 mm Hg.